Cytochromes P-450 from bovine adrenocortical mitochondria will be purified in order to isolate and characterize the enzymes responsible for 11 beta- and 18-hydroxylation of steroids. The methods used will include affinity chromatography. The subunit structures of these enzymes will be determined by electrophoresis on polyacrylamide gels with SDS, for comparison with the behavior of the subunits of the side-chain cleavage P-450 which have already been characterized in this laboratory; it was found that this P-450 can exist in forms composed of 16, 8 or 4 subunits. The subunits of these enzymes will be prepared for amino acid analysis. The size (number of subunits 16, 8 and/or 4) of the active form of the side-chain cleavage enzyme will be determined by delta A340(360) in the analytical ultracentrifuge and by studying the behavior of the enzyme on polyacrylamide gel electrophoresis (without SDS) when it is first mixed with substrate, TPHN and electron transport proteins. The kinetics of the side-chain cleavage of cholesterol and cholesterol SO4 by the purified enzyme will be determined and compared. BIBLIOGRAPHIC REFERENCES: Takagi, Y.; Shikita, M. and Hall, P.F. The Active Form of Cytochrome P-450 from Bovine Adrenocortical Mitochondria. J. Biol. Chem. 250: 8445-8448, 1975. Hall, P.F. and Lipson, E.D. Involvement of Heme in Conversion of 20S, 22R-Dihydroxycholesterol to Pregnenolone Catalysed by Adrenal Cytochrome P-450. Federation Proceedings 34: 567, 1975.